To determine the rates of cellular NAD
+ synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD
+ should be quantified. Although with
2H (D)-labeled precursors [2,4,5,6-D
4]nicotinamide ([D
4]Nam) and [2,4,5,6-D
4]nicotinic acid ([D
4]NA), [D
3]NAD
+ is formed in human cells, why only three of four D atoms from [D
4]Nam and [D
4]NA are present in NAD
+ remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/
1H (H) exchange at the redox site of NAD
+/NADH (C-4 carbon of the pyridine ring) by oxidoreductases exhibiting opposite stereospecificity for the coenzymes in the 1-Da mass decrease in the cellular NAD
+ formation. In all cells examined, [
Nam-D
3]NAD
+, but not [
Nam-D
4]NAD
+, was obtained after the incubation with the D
4-labeled precursors, whereas [
Nam-D
4]NAD
+, but not [
Nam-D
3]NAD
+, was synthesized from the same precursors with purified recombinant NAD
+ biosynthetic enzymes. [D
4]Nam group of [
Nam-D
4]NAD
+ was converted to [D
3]Nam group via [D
4]NADH by in vitro sequential reduction and oxidation with oxidoreductases exhibiting opposite stereospecificity for the coenzymes. Furthermore, using [2,5,6-D
3]Nam, which has H instead of D at the C-4 carbon, as a precursor of NAD
+ in the cells, the 1-Da mass decrease in the nucleotide was not observed. Based on these observations, we conclude that following the synthesis of [
Nam-2,4,5,6-D
4]NAD
+, cellular redox reactions of NAD
+/NADH convert [
Nam-2,4,5,6-D
4]NAD
+ to [
Nam-2,5,6-D
3]NAD
+. Quantification of [
Nam-2,5,6-D
3]NAD
+ and [2,5,6-D
3]Nam would successfully determine the rate of the NAD
+ turnover and provide clues to understand regulatory mechanisms of cellular NAD
+ concentrations.
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